A Novel Method to Generate MNase Ladders Reveal Rapid Chromatin Remodeling upon Gametogenesis and Mating in Chlamydomonas

Publication date: Available online 17 July 2018Source: ProtistAuthor(s): Pooja Potdar, Patricia Pinto, Nicole D’Souza, Prajakta Joshi, Akshay Malwade, Subhojit SenTo circumvent nuclei isolation for nucleosomal mapping of wild-type (cell walled) algal cells, we developed a quick and versatile methodology, by abrasion of whole cells (Chlamydomonas, Scenedesmus and yeast), allowing Micrococcal Nuclease (MNase) direct access to nuclear chromatin, in situ. Varying parameters such as bead abrasion, vortex and incubation conditions, we optimized capture of an ‘early digest’ which may probe chromatin differentially, based on nucleosome accessibility. A comparison of such ladders across vegetative cells, gametes and zygotes revealed an increase in the average nucleosomal repeat length (+17-34 nt) upon gametogenesis, indicating a trend of chromatin compaction. Using PCR, we compared promoter enrichment in increasing orders of fractionated nucleosomal repeats (mono-, di-, up to penta-), each differing in cleavability based on chromatin accessibility. Concordant with higher gene expression (mating locus), promoters revealed an enrichment in mono-nucleosomal fractions. Interestingly, the zygote specific gene, MT0828 displayed rapid remodelling from penta-nucleosomal enrichment when completely repressed (vegetative), to intermediate states during gametogenesis (24 hrs), which finally shifted to being largely mono-nucleosomal, when induced (1 h zygotes). Summarizing three ca...
Source: Protist - Category: Molecular Biology Source Type: research